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Epidermal growth factor receptor trans-activation mediates the tonic and fibrogenic effects of endothelin in the aortic wall of transgenic mice¹

MARTIN FLAMANT, PIERRE-LOUIS THARAUX, SANDRINE PLACIER, DANIEL HENRION, TOM COFFMAN^#, CHRISTOS CHATZIANTONIOU and JEAN-CLAUDE DUSSAULE^*2

INSERM Unité 489, Hôpital Tenon, 75020 Paris;
INSERM Unité 541, Hôpital Lariboisière, 75010 Paris, France;
^# Division of Nephrology, Duke University Medical Center, Durham, North Carolina, USA; and
^* AP-HP, Service de Physiologie, CHU St.-Antoine, 75012 Paris, France

²Correspondence: Pr. Jean-Claude Dussaule, INSERM U. 489, Paris 75020, France. E-mail: jean-claude.dussaule{at}sat.ap-hop-paris.fr

SPECIFIC AIMS

We addressed the hypothesis that activation of the epidermal growth factor (EGF) receptor pathway plays an important role in the profibrotic and contractile actions of endothelin (ET-1). Using two strains of mice and pharmacological blockers of the EGF receptor, we showed that endothelin-induced activation of collagen I gene and its acute pressor effect are mediated by activation of the EGF receptor.

PRINCIPAL FINDINGS

1. EGF receptor as mediator of the ET-1-induced activation of collagen I gene
Experiments were performed in aortas isolated from transgenic mice harboring the luciferase gene under control of the collagen I-2 chain promoter (procol2(I)). The choice of these mice was based on previous studies showing that the expression pattern of the luciferase reporter gene in embryos and adult animals closely correlates with tissue distribution of collagen I under physiological and/or pathological conditions.

Endothelin administration induced an almost twofold increase of luciferase activity in freshly isolated aortas (Fig. 1 , upper). To test whether phosphorylation of the EGF receptor was involved, incubations were carried out in the presence of the specific inhibitor of EGF receptor activation, PD153035. As shown in Fig. 1 (upper panel), PD153035 completely blocked the stimulatory effect of ET-1 on procol2(I) gene. The fibrogenic effect of ET-1 was canceled by bosentan, an ETA/B receptor antagonist.

View larger version (14K): [in this window] [in a new window]   Figure 1. Upper panel: Luciferase activity from aorta isolated from transgenic mice, under control conditions and in the presence of ET-1 (100 nM) with or without the EGFR inhibitor PD153035 (1 nM). Values are means ± SE of 8 independent measurements/condition. *P < 0.05 compared with control; #P < 0.05 compared with ET-1. Lower panel: Luciferase activity in aorta freshly isolated from transgenic mice under control conditions and in the presence of ET-1 (100 nM) with or without the MAPK/ERK inhibitor PD98059 (50 µM). Values are means ± SE of 6 independent measurements/condition. *P < 0.05 compared with control; #P < 0.05 compared with ET-1.

To determine whether the MAPK/ERK pathway was involved in this fibrogenic effect of ET-1, we used PD98059, a specific inhibitor of the MAPK/ERK activation. PD98059 completely blocked the stimulatory effect of ET-1 on procol2(I) (Fig. 1 , lower panel).

2. EGF receptor as mediator of the ET-1-induced activation of the MAPK/ERK
Next we verified whether ET-1 activated the MAPK/ERK cascade in our protocol. Western blot analysis in freshly isolated mouse aortas indicated that ET-1 produced a twofold increase of MAP kinase activity (223±51% vs. control, P<0,01, n=7). This effect was completely blocked by pretreatment with the MAPK/ERK inhibitor PD98059 (50 µM) (90±10%; P<0.01 vs. ET-1).

In additional experiments, this ET-1-induced activation of the MAPK pathway was canceled by the dual ETA/B receptor antagonist bosentan (191±32 vs. 118±45% of baseline for ET-1 alone or in the presence of bosentan, respectively, P<0.05, n=5).

To examine whether EGFR tyrosine kinase was involved in the signaling pathway of ET-1, we tested whether the EGF receptor was phosphorylated after stimulation of aortic segments with ET-1. Immunoblot analysis indicated that ET-1 (100 nM) induced a rapid phosphorylation of the EGF receptor to a level similar to that induced by 50 ng of EGF.

Next we investigated whether the ET-1-induced activation of EGF receptor was involved in the activation of the MAPK pathway. The ET-1-induced activation of MAP kinase (184±51% vs. control, P<0,05, n=6) was canceled when aortic fragments were pretreated with the specific inhibitor of the EGF receptor phosphorylation PD153035 (1 nM) (92±33% for PD153035+ET-1, P<0.05 vs. ET-1, n=6).

3. Involvement of the EGF receptor in ET-1-induced contractility
Addition of ET-1 in aortic ring segments increased vascular wall tension to 35% of the KCl-induced aortic contraction. This constrictor effect was markedly reduced when PD153035 was applied to aortic rings before ET-1 (34±15 vs. 10±10% of the KCl-induced aortic contraction for ET-1 and ET-1+PD153035, respectively, P<0.05, n=6).

4. Effect of EGF receptor inhibition on ET-1-induced systolic pressure increase
This set of experiments tested whether the EGF receptor activation participates in the in vivo ET-1-induced pressor effect. ET-1 alone or mixed with PD153035 or AG1478 was administered i.v. in anesthetized animals. As expected, ET-1 produced a strong and sustained increase of blood pressure (Fig. 2 A, upper); this pressor effect of ET-1 was canceled in the presence of PD153035 (Fig. 2A , middle). Similar recordings were obtained with AG1478. The ET-1-induced increase of systolic pressure averaged 42 ± 12 mmHg above baseline when injected alone (Fig. 2A , lower panel) but was reduced at 6 ± 5 mmHg of when injected in the presence of the EGF receptor inhibitors.

View larger version (19K): [in this window] [in a new window]   Figure 2. A) Upper panel: Representative recording of systolic blood pressure increase after i.v. injections of ET-1 (80 pmol) either alone (upper) or mixed with the EGFR inhibitor PD153035 (6 pmol) (lower). Similar results were obtained when another specific EGFR inhibitor (AG1478) was used instead. Lower panel: Means ± SE of 8 mice/agent. *P < 0.05 compared with control; #P < 0.05 compared with ET-1. B) Upper panel: Representative recording of systolic blood pressure increase after i.v. administration of ET-1 (80 pmol), followed 5 min later by the EGFR inhibitor PD153035 (6 pmol). Lower panel: Means ± SE of 6 experiments. *P < 0.05 compared with control; #P < 0.05 compared with ET-1 administration.

We further investigated the ability of the EGF receptor tyrosine kinase inhibition to reduce the sustained effect of ET-1-induced vasoconstriction. As shown in Fig. 2B , ET-1 increased the systolic pressure from 80 ± 15 to 137 ± 12 mmHg (P<0.01, n=5); this increase was reduced to 84 ± 10 mmHg after injection of PD153035.

To confirm the role of EGF receptor activation as mediator of the ET-1 vasopressor effect, we used waved-2 mice, which express mutant EGF receptors and display reduced response to the EGF-induced tyrosine kinase activity (10% of the control). In anesthetized animals, bolus injection of ET-1 (80 pmol) produced 11 ± 2 mmHg increase above systolic pressure baseline, whereas the same dose of ET-1 increased systolic pressure by 34 ± 17 mmHg in the wild-type control mice (P<0.05, n=5 and 8 for waved-2 and controls, respectively).

CONCLUSIONS AND SIGNIFICANCE

In the present study, using a complementary ex vivo-in vivo approach and two different strains of genetically modified animals, we observed that activation of the EGF receptor mediates the fibrogenic and contractile actions of ET-1. We found that ET-1-induced activation of collagen I gene was dependent of EGF receptor and MAPK activation and that pharmacological inhibition of EGF receptor phosphorylation canceled this fibrogenic effect of ET-1. Pharmacological blockade of the EGF receptor activation blunted the vasoconstrictor action of ET-1 in isolated aortic rings and anesthetized animals. The involvement of the EGF receptor as mediator of the ET-1 pressor response was further confirmed in genetically modified mice expressing EGF receptors with reduced functionality.

Several recent studies have established a major role for ET-1 in the development of vascular fibrosis. In agreement with this notion, we had previously reported that pharmacological blockade of ET-1 action prevented the development of vascular and renal fibrosis during experimental hypertension by inhibiting the activation of collagen I gene. The present studies confirm these earlier results and extend the mechanism of fibrogenic action of ET-1 by implying the involvement of the MAPK pathway. Addition of ET-1 was accompanied by a rapid phosphorylation of MAPK/ERK and an increase of procol1 (2) activity (Fig. 1) ; inversely, inhibition of MAPK/ERK phosphorylation canceled the ET-1-induced activation of collagen I gene (Fig. 1) . An ET-1-induced activation of the MAPK/ERK pathway had been observed in cultured smooth muscle or mesangial cells and in aortic tissue in vitro. Our study is among the first to imply that this activation of the MAPK cascade is involved in the fibrogenic action of ET-1 in the vascular wall.

The interaction between ET-1 and EGF receptor to produce mitogenesis and cell proliferation is well established in the literature. Less known is whether this interaction extends to the trophic effects of ET-1. A novel finding of our study is that the ET-1-EGF receptor–MAPK interaction participates in the fibrogenic effect of ET-1. The pharmacological blockade of EGF receptor phosphorylation canceled the ET-1-induced activation of MAPK and collagen I gene (Fig. 1) . It would be interesting to test whether a similar interaction exists in physiopathological conditions of vascular fibrosis and whether pharmacological blockers of the EGF receptor could be used as anti-fibrotic drugs.

Another important novel finding of our study is that EGF receptor activation is a major mediator of the vasoconstrictor effect of ET-1. This finding was corroborated by three different complementary approaches: pharmacological blockade of the EGF receptor activation completely inhibited the ET-1-induced increase of vascular wall tension in aortas ex vivo (Fig. 2) ; pharmacological blockade of the EGF receptor activation prevented and/or reversed the ET-1-induced blood pressure increase in normal animals in vivo (Fig. 2) ; and the ET-1 pressor effect was substantially reduced in mutant animals displaying malfunctioning EGF receptors. Few studies have examined the role of the EGF receptor in the vasoconstrictor action of peptides such as Ang II or ET-1. A proper vasoconstrictor action of EGF has been observed when aortas were isolated from hypertensive animals in which Ang II or ET-1 play an important role, such as the DOCA/salt, L-NAME, or SHR models. It is possible that the role of the EGF receptor as a mediator of vasoconstriction depends on the physiopathological context of the vascular wall: under normal conditions its involvement seem to be negligible; in contrast, the trans-activation of the EGF receptor appears to become an important factor of vascular contractility in pathologies where the endothelin or the renin-angiotensin systems are activated.

This study provides new information about the mechanisms by which endothelin promotes fibrogenesis and contraction within the vascular wall. The ET-1-induced activation of collagen I gene depends on MAPK activation, due at least partly to EGF receptor trans-activation. The EGF receptor trans-activation appears to be an important factor in ET-1-induced vasoconstriction. Thus, a single cellular phenomenon, EGF receptor activation mediates the acute trophic and vasomotor effects of a potent vasoconstrictor such as endothelin. This observation offers a new perspective about the role of the EGF receptor and the eventual use its inhibitors in cardiovascular or hypertensive pathologies.

View larger version (29K): [in this window] [in a new window]   Figure 3. Schematic diagram depicting the proposed pivotal role EGF receptor plays in mediating the fibrogenic and contractile actions of endothelin in aortas. ET-1 by acting on its receptors trans-activate the EGF receptor. Once the EGF receptor is phosphorylated, it activates the MAP/ER kinase pathway, which in turn induces fibrogenesis by activating synthesis of extracellular matrix proteins such as collagen I. In parallel, the EGF receptor activation participates in ET-1-induced vasoconstriction by yet undefined pathways. Both actions of ET-1 are essential and cooperate to produce remodeling of the vasculature. Red arrows indicate the inhibitory action of the pharmacological tools used in the study.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0115fje; to cite this article, use FASEB J. (December 3, 2002) 10.1096/fj.02-0115fje

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